Such a microscope and/or microscopy method are or is known, for example, from the publication of C. Müller and J. Enderlein, Physical Review Letters, 104, 198101 (2010), or the EP 2317362 A1, which also cites additional references to the prior art.
This approach achieves an increase in resolution by imaging a spot in a diffraction limited manner onto a plane of detection. The diffraction-limited image images a point spot as an Airy disk.
This diffraction disk is detected in the plane of detection in such a way that its structure can be resolved. Based on the imaging performance of the microscope, the result is an oversampling on the part of the detector. When imaging a point spot, the form of the Airy disk is resolved. The resolution can be increased by a factor of two beyond this diffraction limit by suitably evaluating the diffraction pattern, which is described in the aforementioned documents; and the disclosure of these documents is hereby incorporated in their entirety.
At the same time, however, it is unavoidable on the detection side that for each point, which is sampled on the sample in this manner, compared to a conventional laser scanning microscope (hereinafter also referred to by the acronym LSM), a still image has to be captured with a plethora of image data. If the structure of the still image of the spot is detected, for example, with 16 pixels, then each spot would have not only 16 times the amount of data, but also a single pixel would have an average of only 1/16 of the radiation intensity that would fall on the detector of an LSM during a conventional pinhole detection. Since the radiation intensity is, of course, not uniformly distributed throughout the structure of the still image, for example, the Airy disk, the radiation intensity at the edge of this structure is actually much less than the mean value of 1/n for n pixels.
Therefore, one is faced with the problem of the detector being able to achieve a high resolution detection of radiation quantities. Conventional CCD (charge coupled diodes) arrays, which are commonly used in microscopy, do not achieve a sufficient signal-to-noise ratio, so that even an extension of the image acquisition time, which by itself would already be a disadvantage in the application, would not help. APD (avalanche photodiode) arrays are also subject to excessive levels of dark noise, so that even an extension of the measuring time would result in an insufficient signal-to-noise ratio. The same applies to CMOS detectors, which are also disadvantageous with respect to the size of the detector element, because the diffraction-limited still image of the spot would fall on too few pixels. PMT (photo multiplier tube) arrays are also associated with similar design space problems. In this case the pixels are also too large. Therefore, the design space problems are based, in particular, on the fact that a high resolution microscope can be realized in terms of the development effort and the distribution of the device, only if integration into existing LSM designs were possible. In such microscopes, however, specific sizes of the still image are specified. A detector, which is larger in terms of area, could be incorporated, only if it were possible to provide, in addition, an optical system that once again significantly expands the image, i.e., by several orders of magnitude. Such an optical system is expensive and complicated in its design, if the objective is to obtain a diffraction-limited pattern without additional aberrations.
Other methods which avoid the above described problems associated with the high resolution detection are also known from the prior art. For example, the EP 1157297 B1 discloses a method that exploits nonlinear processes by means of structured illumination. A structured illumination is moved across the sample in a plurality of rotational and spatial positions and orientations; and the sample is imaged in these different states on a wide field detector, for which the described limitations do not exist.
A method, which also achieves a high resolution (i.e., a resolution of a sample image beyond the diffraction limit) without the described limitations of the detector, is known from the WO 2006127692 and the DE 102006021317. This method, which is known by the acronym PALM [photo activated localization microscopy], uses a marker substance that can be activated by means of an optical activation signal. Only when the marker substance is in the activated state is it possible for the marker substance to be excited with excitation radiation to emit a certain fluorescence radiation; even when exposed to excitation radiation, non-activated molecules do not emit any fluorescence radiation. Thus, the activation radiation switches the activating substance into a state, in which it can be excited to fluoresce. Therefore, one generally speaks of a switch-over signal. At this point this switch-over signal is applied in such a way that at least a certain proportion of the activated marker molecules are spaced apart from the adjacent marker molecules, which are also activated, in such a way that the activated marker molecules, measured on the basis of the optical resolution of microscopy, are separated or can be subsequently separated. This procedure is referred to as isolating the activated molecules. For these isolated molecules, it is easy to determine the center of their resolution-limited radiation distribution and, based thereon, to computationally determine the location of the molecules with higher accuracy than the optical imaging actually allows. In order to image the entire sample, the PALM method exploits the fact that the probability of a marker molecule being activated by the switch-over signal of a given intensity is the same for all marker molecules. Hence, the intensity of the switch-over signal is applied in such a way that the desired isolation occurs. These process steps are repeated until as many of the marker molecules as possible are included once in a subset that was excited to fluoresce.